Antimicrobial conjugates

ABSTRACT

The present invention presents a metallic nanoparticle-ligand-photosensitiser conjugate, a method of making such conjugate, and a method of using such mixture for killing or preventing the growth of microbes.

CLAIM OF BENEFIT OF FILING DATE

This application claims the benefit of U.S. Provisional PatentApplication Ser. No. 60/821,423 titled: “Antimicrobial Mixtures” filedon Aug. 4, 2006, U.S. Provisional Patent Application Ser. No. 60/868,130titled: “Antimicrobial Conjugates” filed on Dec. 1, 2006, and UnitedKingdom Patent Application No. 0712287.2 titled: “AntimicrobialConjugates” filed on Jun. 22, 2007.

FIELD OF INVENTION

The present invention relates to mixtures comprising charge-stabilizedmetallic nanoparticles and a photosensitiser, and their use as lightactivated antimicrobials. The present invention also relates to metallicnanoparticle-ligand-photosensitiser conjugates and their use as lightactivated antimicrobials.

BACKGROUND OF THE INVENTION

Photosensitisers, such as toluidine blue O, act as light-activatedantimicrobial agents. Although they may have no antimicrobial activityat low concentrations in the dark, when irradiated with light of acertain wavelength (such as 633 nm for toluidine blue O) they are ableto kill a wide range of microbes. Killing is thought to be due to thesinglet oxygen produced on irradiation of the compound. There isconsiderable interest in enhancing the activity of existingphotosensitisers. The present invention focuses on one method ofachieving this.

US 2005/0058713 describes that singlet oxygen production by aphotosensitiser (zinc phthalocyanine) is enhanced by covalently linkingit to gold nanoparticles (see also Duncan C. Hone, Peter I. Walker,Richard Evans-Gowing, Simon FitzGerald, Andrew Beeby, IsabelleChambrier, Michael J. Cook, and David A. Russell. Langmuir 2002, 18,2985-7). However, this increase in singlet oxygen generation has beenreported to be due, at least in part, to the presence oftetraoctylammonium bromide—a reagent used in the preparation of thephthalocyanine-nanogold. The authors concluded, therefore, that thesinglet oxygen generating system was, in fact, a three-component systemconsisting of nanogold, the phthalocyanine and the tetraoctylammoniumbromide. Although the phthalocyanine/nanogold/tetraoctylammonium bromidewas found to increase singlet oxygen generation, it was not demonstratedthat these particles were able to kill either mammalian cells ormicrobes.

Nanoparticle suspensions are inherently unstable, and the nanoparticlestend to associate, or clump together. Two methods are used to counterthis. One is ligand-stabilization, which is employed, for example, in US2005/0058713. The other is charge-stabilization.

The present inventors have found that, surprisingly, simple mixing ofcharge-stabilized metallic nanoparticles with a photosensitiser resultsin enhancement of antimicrobial activity.

The present inventors have also found that, surprisingly, metallicnanoparticle-ligand-photosensitiser conjugates, in which aphotosensitiser is directly bound, via the ligand, to ligand-stabilisednanoparticles, have enhanced antimicrobial properties.

SUMMARY OF THE INVENTION

In one aspect of the invention there is provided a mixture comprisingcharge-stabilized metallic nanoparticles and a photosensitiser. Theinvention also provides a process for preparing such a mixture.

In another aspect, the present invention provides use of the mixtures asantimicrobials.

In yet another aspect, the present invention provides use of themixtures in the manufacture of a medicament for killing or preventingthe growth of microbes.

The present invention also provides a process of killing or preventingthe growth of microbes, comprising using the mixtures of the presentinvention.

In another aspect, the present invention provides use of a metallicnanoparticle-ligand-photosensitiser conjugate, wherein: the ligand is awater-solubilising ligand; and the metallic nanoparticle andphotosensitiser are chosen such that the conjugate generates singletoxygen and/or free radicals as a light-activated antimicrobial.

In one aspect, the use as an antimicrobial is for inanimate objects andsurfaces.

In another aspect, the present invention provides the above-mentionedconjugates for use in killing or preventing the growth of microbes orfor ameliorating or reducing the incidence of proliferative celldisorders such as cancer in the human or animal body.

The present invention also provides new metallicnanoparticle-ligand-photosensitiser conjugates, comprising gold,tiopronin and toluidine blue, and a process for making these and otherconjugates useful in the present invention. Photodisinfection can meetthe need to treat infections and decolonize microbes residing in bodycavities without the use of antibiotics.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the effect of TBO and the TBO-tiopronin-gold nanoparticleconjugate on viability of S. aureus 6571 following exposure to whitelight for 30 minutes, or incubation in the dark with TBO or theTBO-tiopronin-gold nanoparticle conjugate. The white bar (□) in FIG. 1denotes the viable count of the original bacterial suspension, and thedotted bar (

) represents the viable count of the bacterial suspension after exposureto white light alone. The diagonal stripe bar (

) represents the viable count of the bacterial suspension afterincubation in the dark with TBO. The horizontal strip bar (

) represents the viable count of the bacterial suspension afterincubation in the dark with the TBO-tiopronin-gold nanoparticleconjugate. The grey bar (

) represents the viable count of the bacterial suspension after TBO andexposure to white light. The black bar (▪) represents the viable countof the bacterial suspension after TBO-tiopronin-gold nanoparticleconjugate and exposure to white light.

FIG. 2 shows the effect of TBO and the TBO-tiopronin-gold nanoparticleconjugate on viability of S. aureus 6571 following exposure to HeNelaser light for 1 minute, or incubation in the dark with TBO or theTBO-tiopronin-gold nanoparticle conjugate. The white bar (□) in FIG. 2denotes the viable count of the original bacterial suspension, and thedotted bar (

) represents the viable count of the bacterial suspension after exposureto HeNe laser light alone. The diagonal stripe bar (

) represents the viable count of the bacterial suspension afterincubation in the dark with TBO. The horizontal strip bar (

) represents the viable count of the bacterial suspension afterincubation in the dark with the TBO-tiopronin-gold nanoparticleconjugate. The grey bar (

) represents the viable count of the bacterial suspension after TBO andexposure to HeNe laser light. The black bar (▪) represents the viablecount of the bacterial suspension after TBO-tiopronin-gold nanoparticleconjugate and exposure to HeNe laser light.

DESCRIPTION OF THE PREFERRED EMBODIMENT I. NANOPARTICLE-PHOTOSENSITISERMIXTURES

The term “nanoparticles” is generally understood to mean particleshaving a diameter of from about 1 to about 100 nm. Preferably, thenanoparticles used in the present invention have a diameter of fromabout 1 to about 30 nm. In one embodiment, the nanoparticles preferablyhave a diameter of from about 2 to about 5 nm. In another embodiment,the nanoparticles preferably have a diameter of from about 10 to about25 nm, more preferably about 15 to about 20 nm.

Nanoparticles typically, but not exclusively, comprise metals. They mayalso comprise alloys of two or more metals, or more complex structuressuch as core-shell particles, rods, stars, spheres or sheets. Acore-shell particle may typically comprise a core of one substance, suchas a metal or metal oxide or silica, surrounded by a shell of anothersubstance, such as a metal, metal oxide or metal selenide. The term“metallic” as used herein is intended to encompass all such structureshaving a metallic outer surface.

In a preferred embodiment, the outer surface of the metallicnanoparticles of the present invention comprises a main group metal ortransition metal, such as cobalt. More preferably, the metallicnanoparticles are gold, silver or copper nanoparticles, or alloys of twoor more of these metals. Most preferably, the nanoparticles are goldnanoparticles.

A photosensitiser is a compound that can be excited by light of aspecific wavelength. Thus, such a compound may have an absorption bandin the ultraviolet, visible or infrared portion of the electromagneticspectrum and, when the compound absorbs radiation within that band, itgenerates cytotoxic species, thereby exerting an antimicrobial effect.The effect may be due to creation of singlet oxygen but the invention isnot limited to photosensitisers that exhibit antimicrobial effectsthrough creation of singlet oxygen.

Without wishing to be bound by theory, it is thought that thephotosensitiser and nanoparticles are associated via dative covalentbonds, wherein the electrons are provided by, for example, S or Nmoieties on the photosensitiser.

Any photosensitiser may be used in the present invention. However, it ispreferable that the photosensitiser is non-toxic to humans and animalsat the concentrations employed in the present invention. It is alsopreferable that the photosensitiser demonstrates antimicrobial activitywhen exposed to visible light. The photosensitiser is suitably chosenfrom porphyrins (e.g. haematoporphyrin derivatives, deuteroporphyrin),phthalocyanines (e.g. zinc, silicon and aluminium phthalocyanines),chlorins (e.g. tin chlorin e6, poly-lysine derivatives of tin chlorine6, m-tetrahydroxyphenyl chlorin, benzoporphyrin derivatives, tinetiopurpurin), bacteriochlorins, phenothiaziniums (e.g. toluidine blueO, methylene blue, dimethylmethylene blue), phenazines (e.g. neutralred), acridines (e.g. acriflavine, proflavin, acridine orange,aminacrine), texaphyrins, cyanines (e.g. merocyanine 540), anthracyclins(e.g. adriamycin and epirubicin), pheophorbides, sapphyrins, fullerene,halogenated xanthenes (e.g. rose bengal), perylenequinonoid pigments(e.g. hypericin, hypocrellin), gilvocarcins, terthiophenes,benzophenanthridines, psoralens and riboflavin. Other possibilities arearianor steel blue, tryptan blue, crystal violet, azure blue cert, azureB chloride, azure 2, azure A chloride, azure B tetrafluoroborate,thionin, azure A eosinate, azure B eosinate, azure mix sicc. and azureII eosinate.

In one embodiment, particularly preferred photosensitisers are toluidineblue O, methylene blue, dihaematoporphyrin ester, tin chlorin e6,indocyanine green or nile blue sulphate. More preferably, thephotosensitiser is toluidine blue O, methylene blue or tin chlorin e6.Most preferably, the photosensitiser is methylene blue or toluidine blueO.

In a particularly preferred embodiment, the mixture comprises goldnanoparticles and methylene blue or toluidine blue O.

A. Process for Preparation of the Mixtures

In one embodiment, the mixtures of the present invention are in the formof a solution. Such a solution may be produced by contacting a solutionof charge-stabilized metallic nanoparticles with a solution ofphotosensitiser. The mixtures are contacted at any suitable temperature,for example between the freezing point and boiling point of the solventemployed (or at a temperature at which both solutions are liquid ifdifferent solvents are employed). However, if the temperature is toohigh, the nanoparticle solution may become unstable. It is preferredthat the nanoparticle solution remains in a stable condition. In oneembodiment, the solutions are contacted at or about room temperature.

In one embodiment, a solution of metallic nanoparticles is mixed with asolution of photosensitiser and allowed to stand at room temperature forat least about 10 minutes, preferably between about 10 minutes and about1 hour, more preferably between about 15 and about 20 minutes.

Typically, the metallic nanoparticle solution and/or the photosensitisersolution is a solution in a polar solvent, preferably an aqueoussolution, such as in water or phosphate buffered saline solution, inparticular in a pharmaceutically acceptable aqueous carrier. Morepreferably, both the nanoparticle and photosensitiser solutions areaqueous.

The pH of solutions may be such that no adjustment is required uponmixing, or the pH of the mixture may be controlled by the use of asuitable buffer. For example, when the mixture is to be applied to thebody, the pH of the mixture should not be outside the physiological pHrange for the site. The physiological pH range depends on the site inquestion, e.g. intact skin can have a pH as low as 4.2.

The two solutions may be mixed in any proportion, such that the desiredconcentration is achieved in the mixed solution. In one embodiment, theinitial concentrations of each solution are selected as required so thatthe desired concentration in the mixed solution is achieved when equalvolumes of metallic nanoparticle solution and photosensitiser solutionare mixed together.

The desired concentration of the nanoparticles in the mixture depends onthe desired final concentration at the site to be treated. This may varyand a suitable choice depends both on the size of the nanoparticle andthe concentration of the photosensitiser solution. The finalconcentration of the nanoparticles in the mixture is preferably fromabout 1×10¹¹ to about 5×10¹⁵ particles/ml, more preferably from about3×10¹¹ to about 1×10¹⁵ particles/ml. In order to obtain such a finalconcentration, the initial concentration of the nanoparticle solution istypically from about 1×10¹² to about 1×10¹⁶ particles/ml. If thenanoparticle solution as prepared, or as obtained commercially, is ofhigher concentration than this, it may be necessary to dilute thenanoparticle solution before mixing with the photosensitiser. Forexample, an original nanoparticle solution containing 1×10¹⁴ or 1×10¹⁵particles/ml may be diluted 1:10 to 1:100, such that the concentrationbefore mixing with the photosensitiser solution is from 1×10¹² to1×10¹⁴.

The initial concentration of photosensitiser solution is preferablychosen such that when mixed with the nanoparticle solution, the finalconcentration of photosensitiser at the treatment site is from about 5to about 100 μM, more preferably from about 20 to about 50 μM.

It should be noted that the final concentration at the treatment sitemay not necessarily correspond to the concentration in the mixedsolution. For instance in the treatment of periodontal pockets andwounds the treatment site may be flooded with body fluid such as salivaor blood. In such cases, it may therefore be necessary to apply thenanoparticle-photosensitiser mixture in greater concentration so as toachieve an effective concentration after dilution by the body fluid.

B. Antimicrobial Effect of the Mixtures

The mixtures of the present invention have an antimicrobial effect, i.e.they are capable of killing or inhibiting the growth of microorganisms,including bacteria, viruses, fungi and prions, that can cause disease inhumans, animals or plants. In one embodiment, the mixtures of thepresent invention are used to kill or inhibit the growth ofStaphylococcus aureus. Staphylococcus aureus as used in this applicationshall also include Methicillin-Resistant Staphylococcus aureus (“MRSA”).The mixtures of the present invention may also be used to kill orinhibit the growth of Propionibacterium acnes.

In another embodiment, the mixtures of the present invention are used tokill or prevent the growth of the microbes involved in oral diseases,such as inflammatory periodontal disease and caries, or in woundinfections and in disinfecting or sterilising wounds and other lesionsin the oral cavity. Thus, the mixtures of the present invention may beused to kill or inhibit the growth of Streptococcus sanguis,Porphyromonas gingivalis, Fusobacterium nulceatum, Actinobacillusactinomycetemcomitans, Candida albicans, Streptococcus mutans andlactobacilli.

The antimicrobial effect of the mixtures is activated by exposure to alight source. In one embodiment, the mixture may be exposed to a lightsource comprising radiation having a wavelength, or a range ofwavelengths, within the range of wavelengths absorbed by thephotosensitiser, preferably near or corresponding to the wavelength ofmaximum absorption of the photosensitiser (λ_(max) ). As describedabove, it is preferred that the photosensitiser demonstratesantimicrobial activity when exposed to visible light, i.e. λ_(max) isbetween about 380 and about 780 nm. For example, toluidine blue Odemonstrates antimicrobial activity when irradiated with light having awavelength of 633 nm.

In general, any light source that emits light of an appropriatewavelength may be used. The source of light may be any device orbiological system able to generate monochromatic or polychromatic light,coherent or incoherent light, especially visible white light. Examplesinclude a fluorescent light source, laser, light emitting diode, arclamp, halogen lamp, incandescent lamp or an emitter of bioluminescenceor chemiluminescence. In certain circumstances, sunlight may besuitable. Preferably, the wavelength of the light emitted by the lightsource may be from about 200 to about 1060 μm, preferably from about 380to about 780 nm. A suitable laser may have a power of from about 1 toabout 100 W. Other suitable lasers may have a power of about 1 to about1000 mW and a beam diameter of from about 1 to about 10 mm. The lightdose for laser irradiation is suitably from about 5 to about 333 J cm⁻²,preferably from about 5 to about 30 J cm⁻² for laser light. For whitelight irradiation, a suitable dose is from about 0.01 to about 100J/cm², preferably from about 0.1 to about 20 J/cm², more preferably fromabout 3 to about 10 J/cm². In a preferred embodiment, the mixture maysuitably be irradiated using a source of white light.

Without limitations, the following are examples of light sources andtheir respective exemplary wavelengths and/or power outputs that may besuitable for use in the present invention:

-   -   Helium neon (HeNe) gas laser (e.g. 633 nm)    -   Argon-pumped dye laser (e.g. 500-700 nm, 5 W output)    -   Copper vapour-pumped dye laser (e.g. 600-800 nm)    -   Excimer-pumped dye laser (e.g. 400-700 nm)    -   Gold vapour laser (e.g. 628 nm, 10 W output)    -   Tunable solid state laser (e.g. 532-1060 nm), including Sd:YAG    -   Light emitting diode (LED) (e.g. 400-800 nm)    -   Diode laser (e.g. 630-850 nm, 25 W output), e.g. gallium        selenium arsenide    -   Tungsten filament lamp    -   Halogen cold light source    -   Fluorescent lamp (e.g. 10 to 30 W)

The present invention is not limited to the above-mentioned examples oflight sources, exemplary wavelengths and/or power outputs. It isentirely possible for the present invention to be carried out usingother light sources and/or the above-mentioned light sources withdifferent wavelengths and/or power outputs.

The duration of exposure to the light source should be long enough toensure sufficient killing. This may vary depending on the choice ofphotosensitiser and light source. For example, toluidine blue O mayrequire exposure for between 10 and 30 minutes to ensure effectivekilling of microbes using a 15 to 30 W fluorescent lamp, but only 20 to60 seconds using a fibre optic white light source. Otherphotosensitisers, such as tin chlorin e6, may require 10 to 30 minuteswith a fibre optic white light source. In one embodiment, the durationof irradiation is suitably from about one second to about 15 minutes,preferably from about 1 to about 5 minutes. In another embodiment, forexample when the light source is of low intensity such as exposure tonatural daylight, the mixture is exposed to the light source for alonger period of time, such as for several hours, for example from about1 to about 12 hours.

The light may be delivered to the mixture by ambient exposure, or, ifnecessary or convenient, by use of a directed means such as a fibreoptic light source or other known optical devices.

The efficacy of the mixtures as antimicrobials depends on many factors.The choice of nanoparticle type, choice of photosensitiser, nanoparticlesize, concentration of nanoparticles and concentration ofphotosensitiser may all influence antimicrobial activity. Thusindividual combinations may have particularly advantageous effects. Forexample and without limitations, the following combinations have beenfound particularly effective against Staphylococcus aureus:

-   -   2 nm diameter gold nanoparticles at a concentration of 4×10¹³    -   particles/ml with toluidine blue O at a concentration of 20 μM.    -   15 nm diameter gold nanoparticles at a concentration of 1×10¹⁴        to 1×10¹⁵ particles/ml with toluidine blue O at a concentration        of 20 to 50 μM.    -   2 nm diameter gold nanoparticles at a concentration of4×10¹¹ to        4×10¹³ particles/ml with methylene blue at a concentration of 20        μM.    -   b 15 nm diameter gold nanoparticles at a concentration of 1×10¹³        to 1×10¹⁵ particles/ml with methylene blue at a concentration of        20 μM.    -   2 nm diameter gold nanoparticles at a concentration of 4×10¹¹        particles/ml with tin chlorin e6 at a concentration of 20 μg/ml.    -   2 nm gold nanoparticles at a concentration of 4×10¹³        particles/ml with nile blue sulphate at a concentration of 20 to        50 μM.        C. Applications of Mixtures

The antimicrobial properties of the mixtures of the present inventionmay find application in hospitals and other places where microbiologicalcleanliness is necessary, for example food processing facilities, diningareas or play areas. Use in abattoirs is also envisaged. The mixturesmay be applied to any suitable surface in order to sterilize it, forexample work surfaces, wash basins, toilets, tiles, door handles orcomputer keyboards. In another embodiment, the mixture may be applied tocling-film or other films or packaging, such as food packaging, forexample by spraying or painting a solution of the mixture onto the film.Such cling-film type material could be wrapped around or used to covermedical/dental instruments, computer input devices, surfaces etc.

The mixtures may be applied as a coating by painting, spreading orspraying and may be dried or allowed to dry naturally. They can also bemixed with a plastics material such as cellulose acetate to create anantimicrobial plastic. Such a plastics material could be used tomanufacture articles, such as computer input devices, or asantimicrobial coverings to be wrapped or coated over the surface of thearticle to be treated. Thus, in one embodiment, an article such as acomputer input device could be coated with a mixture of celluloseacetate, photosensitiser and nanoparticles.

In another embodiment, the antimicrobial properties of the mixtures ofthe present invention may find application in killing the microbesinvolved in oral diseases, as mentioned above. The mixtures of thepresent invention may also find use in killing or preventing the growthof microbes in various body cavities. Body cavity shall mean any cavitywithin a body such as mouth or oral cavity, nose, ear, vagina, lung, theentire digestive tract (e.g., throat, esophagus, stomach, intestines,rectum, etc.), gall bladder, bladder, any open wound or the like. Thebody cavity can be within a human body or a body of another animal.

The mixtures of the present invention may also be applied topically, forexample to the skin, wounds or a mucosal surface in order to kill orprevent the growth of microbes. As a further example, the mixtures ofthe present invention may find application in killing or preventing thegrowth of fungi, for example in infections of the nail bed.

For such applications, the mixture is suitably in the form of a solutionor a suspension in a pharmaceutically acceptable aqueous carrier, butmay be in the form of a solid such as a powder or a gel, an ointment ora cream. The composition may be applied to the infected area bypainting, spreading, spraying, injecting or any other conventionaltechnique.

The present invention also provides use of a mixture of the presentinvention in the manufacture of a medicament for killing or preventingthe growth of microbes, and a method of disinfecting or sterilising alocus in subject, which method comprises the administration to the saidlocus of an effective amount of a mixture of the present inventionfollowed by exposure of said locus to a light source.

In a preferred aspect the invention provides the use of a mixture of thepresent invention in the manufacture of a medicament for use indisinfecting or sterilising tissues of a body cavity or a wound orlesion in a body cavity by (a) contacting the tissues, wound or lesionwith mixture and (b) irradiating the tissues, wound or lesion with lightat a wavelength absorbed by the photosensitiser.

The wound or lesion treated may be any surgical or trauma-induced wound,a lesion caused by a disease-related microbe, or a wound or lesioninfected with such a microbe. The treatment may be applied to disinfector sterilise a wound or lesion as a routine precaution against infectionor as a specific treatment of an already diagnosed infection of a woundor lesion. In one embodiment, the body cavity is the oral cavity. Themixtures of the present invention may also be used in other bodycavities, such as the nose, rectum, vagina, etc.

In another preferred aspect the invention provides the use of a mixtureof the present invention in the manufacture of a medicament for use inkilling or preventing the growth of disease-related microbes in a bodycavity, such as the oral cavity, nose, rectum, vagina, etc. by (a)contacting the microbes with mixture and (b) irradiating the microbeswith light at a wavelength absorbed by the photosensitiser.

When the body cavity is the oral cavity, the treatment with mixture andirradiation are preferably applied to (i) destruction of disease-relatedmicrobes in a periodontal pocket in order to treat chronicperiodontitis; (ii) destruction of disease-related microbes in theregion between the tooth and gingiva (gingival crevice or gingivalmargin) in order to treat or prevent inflammatory periodontal diseases,including chronic periodontitis, gingivitis and the like; (iii)disinfection or sterilisation of drilled-out carious lesions prior tofilling; (iv) destruction of cariogenic microbes on a tooth surface inorder to prevent dental caries; (v) disinfection or sterilisation ofdental and/or gingival tissues in other dental surgical procedures and(vi) treatment of oral candidiasis in AIDS patients, immunocompromisedpatients or patients with denture stomatitis.

For the above applications, the mixture is suitably used in the form ofa pharmaceutical composition comprising the nanoparticles andphotosensitiser in solution in a pharmaceutically acceptable aqueouscarrier. The pharmaceutical composition may further comprise one or moreaccessory ingredients selected from buffers, salts for adjusting thetonicity of the solution, antioxidants, preservatives, gelling agentsand remineralisation agents.

In another aspect, the present invention provides a process of killingor preventing the growth of microbes, comprising contacting with amixture according the present invention followed by exposure to a lightsource for a sufficient amount of time to kill or prevent the growth ofmicrobes. As described above, the mixture is at a suitable concentrationsuch that a desired level of antimicrobial activity is achieved at thetreatment site. Thus, the “final concentrations” as described above arepreferred. For application to surfaces, the mixture may be applieddirectly by any suitable means, such as a cloth, spray or wash. For oralor topical applications, any of the methods mentioned above, i.e.painting, spreading, spraying, injecting or any other conventionaltechnique, may be used to contact the mixture with the microbes.

The mixture may be left in contact with the microbes for a period oftime. This duration of time may vary depending on the particularphotosensitiser in use and the target microbes to be killed. Forexample, it can be from about 1 second to about 10 minutes. In oneembodiment, the duration of time is about 10 seconds to about 2 minutes.In another embodiment, the duration of time is about 30 seconds.

In one aspect, the present invention does not extend to the use of themixtures in methods of treatment of the human or animal body by surgeryor therapy, or in methods of diagnosis conducted on the human or animalbody.

II. METALLIC NANOPARTICLE-LIGAND-PHOTOSENSITISER CONJUGATES

The term “nanoparticle” is generally understood to mean particles havinga diameter of from about 1 to about 100 nm. Preferably, thenanoparticles used in the present invention have a diameter of fromabout 1 to about 30 nm, preferably about 1 to about 20 nm.

Nanoparticles typically, but not exclusively, comprise metals. They mayalso comprise alloys of two or more metals, or more complex structuressuch as core-shell particles, rods, stars, spheres or sheets. Acore-shell particle may typically comprise a core of one substance, suchas a metal or metal oxide or silica, surrounded by a shell of anothersubstance, such as a metal, metal oxide or metal selenide. The term“metallic” as used herein is intended to encompass all such structureshaving a metallic outer surface.

The metallic nanoparticles of the present invention should be chosensuch that, when attached via the ligand to the photosensitiser to formthe conjugate, the conjugate generates singlet oxygen and/or freeradicals. Preferably, the conjugate generates both singlet oxygen andfree radicals.

Singlet oxygen generation may be measured by assay: several such methodsare known to those skilled in the art, for example, photoluminescence.Free radical generation may be measured using electron proton resonance(EPR).

Examples of metallic nanoparticles that may be suitable arenanoparticles having a diameter of greater than about 2 nm which exhibitplasmon resonance in the wavelength band of about 200 to about 1600 nm,i.e. covering the visible to near infrared bands. The plasmon resonancemay be measured by UV spectroscopy. It may be seen for both the free andconjugated nanoparticle. For antimicrobial applications, preferablenanoparticles will exhibit plasmon resonance at wavelengths of fromabout 500 to about 600 nm. Gold nanoparticles, for example, exhibitplasmon resonance in this range.

Another property which may be used to help select a suitablenanoparticle is the molar extinction coefficient of the conjugatedphotosensitiser. When a photosensitiser is conjugated via a ligand to asuitable nanoparticle, the extinction coefficient of the photosensitisermay be enhanced, compared to the extinction coefficient that would beexpected based on an equivalent concentration of the photosensitiseralone. Without wishing to be bound by theory, it is thought that thisenhancement occurs because the photosensitiser coordinates to thesurface of the nanoparticle. Thus, in order to select suitablenanoparticles, the extinction coefficient of the conjugate could bemeasured, using a spectrophotometer. Any enhancement is acceptable.Typically, the extinction coefficient may range anywhere from about 2 toabout 30 times or more; from about 5 to about 30 times or more; fromabout 10 to about 30 times or more and from about 20 to about 30 timesor more, compared to what is expected based on the same concentration ofthe unconjugated photosensitiser.

In a preferred embodiment, the outer surface of the nanoparticles of thepresent invention comprises gold, silver or copper. More preferably, thenanoparticles comprise gold, silver or copper, or alloys of two or moreof these metals, such as gold/silver, gold/copper or gold/silver/copper.Suitable alloys may also contain other metals, such asgold/silver/aluminium.

In another embodiment, the nanoparticles described in the precedingparagraph comprise core-shell particles. It is possible for suchcore-shell particles to comprise a magnetic core or magnetic layer. Anexample of such a magnetic core-shell particle is a particle having amagnetic core and an outer shell which comprises gold. Most preferably,the nanoparticles are gold nanoparticles.

The ligand of the metallic nanoparticle-ligand-photosensitiser conjugateis desired to be a water-solubilising ligand. This means that theconjugate as a whole is water soluble at a concentration of at leastabout 1×10⁻⁸ M (mol dm⁻³) at room temperature (25° C.). Preferably, theconjugate is water soluble at a concentration of at least about 1×10⁻⁷M, more preferably at least about 1×10⁻⁶ M.

The concentration for determining water solubility may be measured byany appropriate method. Suitable methods include UV absorption,inductively coupled plasma mass spectrometry (ICP-MS), SQUID(superconducting quantum interference device) magnetometry, EPR or Ramanspectroscopy.

Examples of suitable ligands are water-solubilising ligands chosen fromsulfur ligands, such as thiols (alkanethiols and aromatic thiols),xanthates, disulfides, dithiols, trithiols, thioethers, polythioethers,tetradentate thioethers, thioaldehydes, thioketones, thion acids, thionesters, thioamides, thioacyl halides, sulfoxides, sulfenic acids,sulfenyl halides, isothiocyanates, isothioureas or dithiocarbamates;selenium ligands, such as selenols (aliphatic or aromatic), selenides,diselenides, dialkyl-diselenides (for example octaneselenol-nanoparticleis obtained from dioctyl-diselenide), selenoxides, selenic acids orselenyl halides; tellurium ligands, such as tellurols (aliphatic oraromatic), tellurides or ditellurides; phosphorus ligands, such asphosphines or phosphine oxides; nitrogen ligands, such as alkanolaminesor aminoacids; and other ligands such as carboxylate ligands (e.g.myristate), isocyanide, acetone and iodine.

Examples of preferred water-solubilising ligands are 3-mercaptopropionicacid, 4-mercaptobutyric acid, 3-mercapto-1,2-propanediol, cysteine,methionine, thiomalate, 2-mercaptobenzoic acid, 3-mercaptobenzoic acid,4-mercaptobenzoic acid, tiopronin, selenomethionine,1-thio-beta-D-glucose, glutathione and ITCAE pentapeptide.

A photosensitiser is a compound that can be excited by light of aspecific wavelength. Thus, such a compound may have an absorption bandin the ultraviolet, visible or infrared portion of the electromagneticspectrum and, when the compound absorbs radiation within that band, itgenerates cytotoxic species, thereby exerting an antimicrobial effect.The effect may be due to creation of singlet oxygen but the invention isnot limited to photosensitisers that exhibit antimicrobial effectsthrough creation of singlet oxygen. In particular, the photosensitisermay generate free radicals, instead of, or as well as, generatingsinglet oxygen.

It is a requirement of the present invention that the photosensitiser ischosen such that, when attached to the metallic nanoparticle-ligand coreto form the conjugate, the conjugate generates singlet oxygen and/orfree radicals. Preferably, the conjugated photosensitiser generates bothsinglet oxygen and free radicals. Singlet oxygen and free radicalgeneration may be measured as described above.

It is preferable that the photosensitiser is non-toxic to humans andanimals at the concentrations employed in the present invention. It isalso preferable that the photosensitiser demonstrates antimicrobialactivity when exposed to visible light. The photosensitiser is suitablychosen from porphyrins (e.g. haematoporphyrin derivatives,deuteroporphyrin), phthalocyanines (e.g. zinc, silicon and aluminiumphthalocyanines), chlorins (e.g. tin chlorin e6, poly-lysine derivativesof tin chlorin e6, m-tetrahydroxyphenyl chlorin, benzoporphyrinderivatives, tin etiopurpurin), bacteriochlorins, phenothiaziniums (e.g.toluidine blue O, methylene blue, dimethylmethylene blue), phenazines(e.g. neutral red), acridines (e.g. acriflavine, proflavin, acridineorange, aminacrine), texaphyrins, cyanines (e.g. merocyanine 540),anthracyclins (e.g. adriamycin and epirubicin), pheophorbides,sapphyrins, fullerene, halogenated xanthenes (e.g. rose bengal),perylenequinonoid pigments (e.g. hypericin, hypocrellin), gilvocarcins,terthiophenes, benzophenanthridines, psoralens and riboflavin. Otherpossibilities are indocyanine green, nile blue sulphate, arianor steelblue, tryptan blue, crystal violet, azure blue cert, azure B chloride,azure 2, azure A chloride, azure B tetrafluoroborate, thionin, azure Aeosinate, azure B eosinate, azure mix sicc. and azure II eosinate.

In one embodiment, particularly preferred photosensitisers are toluidineblue O (TBO), methylene blue, tin chlorin e6, indocyanine green or nileblue sulphate. Preferably, the photosensitiser is not a porphyrin. Morepreferably, the photosensitiser is toluidine blue O, methylene blue ortin chlorin e6. Most preferably, the photosensitiser is methylene blueor TBO.

The proportion of metallic nanoparticle:ligand:photosensitiser may vary.Typically, the nanoparticle comprises many atoms, only some of whichhave ligand molecules covalently bonded thereto. The number ofphotosensitiser molecules attached to each nanoparticle-ligand core mayalso vary. Typically, only some of the ligand molecules will have aphotosensitiser molecule attached. For example, a preferred conjugateaccording to the present invention could have the compositionAu₂₀₁Tiopronin85TBO₉, Au₂₀₁Tiopronin₈₅TBO₁₁ or Au₂₀₁Tiopronin₈₅TBO₁₅.

The conjugate may also comprise further components. For example, it mayhave a targeting moiety associated with it. The targeting moiety can beassociated with the conjugate via any suitable means, for example it maybe attached to the nanoparticle core, to the ligand or to thephotosensitiser. Such targeting moieties may be suitable, for example,for targeting specific microorganisms, or for targeting cancer cells.For example, they may be antibodies with specificity for the targetorganism or cancer cell. Other examples of targeting moieties includebacteriophages, protein A (targets Staphylococcus aureus) and bacterialcell-wall binding proteins or peptides.

The preferred conjugate mentioned above is an example of another aspectof the present invention. Thus the present invention also provides novelmetallic nanoparticle-ligand-photosensitiser conjugates, wherein themetallic nanoparticle comprises gold, the ligand comprises tiopronin andthe photosensitiser comprises (TBO). In one embodiment, the novelconjugate preferably consists of gold-tiopronin-TBO. Preferably, thenovel conjugate comprises from about 5 to about 20 TBO groups pernanoparticle-ligand core.

The novel conjugates of the present invention have been found todemonstrate particularly effective antimicrobial properties. Thus alluses of conjugates as described herein apply to the novel conjugates.

A. Process for Preparation of the Conjugates

The present invention provides a process for producing conjugates asdescribed above. Such a process comprises the steps of:

-   (i) providing a nanoparticle-ligand core, comprising a nanoparticle    having bonded thereto at least one ligand having first and second    functional groups, wherein the ligand is bonded to the nanoparticle    via the first functional group, and then-   (ii) reacting the second functional group of at least one of said    ligands with a functional group of a photosensitiser.

Preferred nanoparticles, ligands and photosensitisers for use in theprocess of the present invention are as described above. Preferably,both steps of the process are carried out in aqueous solution.

One embodiment of the process will now be illustrated by reference tothe novel gold-tiopronin-TBO conjugates described above.

Typically, the nanoparticle-ligand core is prepared by a reaction basedon the Brust reaction (Brust, M; Walker, M; Bethell, D; Schiffrin, D J;Whyman, R; J. Chem. Soc. Chem. Comm., 1994, 801-802). Such reactions arewell known to those skilled in the art. However, in the case of agold-tiopronin core, it is preferable to modify the usual reactionmixture, and the reaction is preferably executed in a methanol/aceticacid mixture, rather than in toluene. Furthermore, the amount of aceticacid should be controlled such that a final pH of about 5 is achievedafter addition of sodium tetrahydroborate.

The nanoparticle-ligand core is preferably purified, for example bydialysis, before reaction with the photosensitiser.

Typically, the reaction between the nanoparticle-ligand core andphotosensitiser takes place in an aqueous medium. In one embodiment, acatalyst can be used. For example,1-[3-(dimethylamino)-propyl]-3]ethyl-carbodiimide (EDC) can be used tocatalyse reactions between tiopronin carboxylic acid groups and anaromatic amine-containing TBO molecule. N-hydroxysulfosuccinimide sodiumsalt may be included in the reaction mixture to improve the efficiencyof the reaction.

Typically, the reaction feed ratio of photosensitiser tonanoparticle-ligand core is such that it provides from about 0.5 toabout 2 functional groups on the photosensitiser per “second functionalgroup” on the ligand. Preferably, the ratio is about 1:1. Such a ratioprovides conjugates with from about 5 to about 20 molecules ofphotosensitiser per core, as described above.

Conjugates prepared by a process according to the present invention aretypically stable, showing no decomposition over a period of months.

B. Conjugate Compositions

Compositions comprising a conjugate for use in the present inventiontypically comprise a solution or suspension of the conjugate in asuitable solvent, such as water or phosphate buffer solution. Asdescribed above, the conjugate as a whole is water soluble at aconcentration of at least about 1×10⁻⁸ M (mol dm⁻³). However, atconcentrations above this lower limit, it is not necessary that theconjugate is completely soluble in water. The conjugate may form asuspension in water, or may be dissolved to form a solution in a mediumwith a higher dielectric constant, such as saline or phosphate bufferedsaline (PBS). Gold-tiopronin-TBO conjugates, for example, may besuspended in water, but are soluble in PBS: the water solubility isdetermined by the TBO content, with lower TBO amounts leading to greaterwater-solubility of the conjugates.

Suitable concentrations of conjugate in a suspension/solution may becalculated based on the amount of photosensitiser present. Thus, forexample a TBO-containing conjugate could be used such that the final TBOcontent is between about 0.01 and about 1 μM, preferably from about 0.1to about 0.5 μM. In another embodiment, the final TBO content ispreferably from about 0.25 to about 5 μM, preferably from about 0.5 toabout 2 μM. The final desired concentration of photosensitiser should besuch that the composition has antimicrobial activity when exposed to alight source, as described further below. The actual concentration willdepend on many factors, including the type of photosensitiser, the lightsource to be used and the duration of exposure. However, without wishingto be bound by theory, it can be generally stated that if theconcentration of photosensitiser is too low, antimicrobial activity maynot be seen due to insufficient generation of singlet oxygen and/or freeradicals, and if the concentration is too high, light penetration intothe solution or suspension may be compromised. In the latter case, anyantimicrobial effect will be suppressed due to failure of much of thecomposition to become “light activated”.

It should be noted that the final concentration of conjugate at a siteto be disinfected may not necessarily correspond to the concentration inthe solution/suspension. For instance in the treatment of periodontalpockets and wounds the treatment site may be flooded with body fluidsuch as saliva or blood. It may therefore be necessary to apply theconjugate composition in greater concentration so as to achieve aneffective concentration after dilution by other fluids, such as bodyfluid and the like.

The pH of solutions may be such that no adjustment is required, or thepH of the composition may be controlled by the use of a suitable buffer.For example, when the composition is to be applied to the body, the pHof the composition is preferably not outside the physiological pH rangefor the site. The physiological pH range depends on the site inquestion, e.g. intact skin can have a pH as low as about 4.2 (Microbialinhabitants of humans: their ecology and role in health and disease.Wilson M (2005) Cambridge University Press).

C. Light Activation

The antimicrobial effect of the conjugates is activated by exposure to alight source. In one embodiment, the conjugates may be exposed to alight source comprising radiation having a wavelength, or a range ofwavelengths, within the range of wavelengths absorbed by the conjugatedphotosensitiser, preferably near or corresponding to the wavelength ofmaximum absorption of the photosensitiser (λ_(max)). In one embodiment,it is preferred that the conjugate demonstrates antimicrobial activitywhen exposed to visible light, i.e. λ_(max) is between about 380 andabout 780 nm.

In general, any light source that emits light of an appropriatewavelength may be used. The source of light may be any device orbiological system able to generate monochromatic or polychromatic light,coherent or incoherent light, especially visible white light. Examplesinclude a fluorescent light source, laser, one or more light emittingdiodes (LEDs), arc lamp, halogen lamp, incandescent lamp or an emitterof bioluminescence or chemiluminescence. In certain circumstances,sunlight may be suitable. Preferably, the wavelength of the lightemitted by the light source may be from about 200 to about 1060 nm,preferably from about 380 to about 780 nm. A suitable laser may have apower of from about 1 to about 100 W. Other suitable lasers may have apower of about 1 to about 1000 mW and a beam diameter of from about 1 toabout 10 mm. The light dose for laser irradiation is suitably from about5 to about 333 J cm⁻², preferably from about 5 to about 30 J cm⁻² forlaser light. For white light irradiation, a suitable dose is from about0.01 to about 100 J/cm², preferably from about 0.1 to about 20 J/cm²,more preferably from about 3 to about 10 J/cm². In a preferredembodiment, the mixture may suitably be irradiated using a source ofwhite light.

Without limitations, the following are examples of light sources andtheir respective exemplary wavelengths and/or power outputs that may besuitable for use in the present invention:

-   -   Helium neon (HeNe) gas laser (e.g. 633 nm, 35 mW output)    -   Argon-pumped dye laser (e.g. 500-700 nm, 5 W output)    -   Copper vapour-pumped dye laser (e.g. 600-800 nm)    -   Excimer-pumped dye laser (e.g. 400-700 nm)    -   Gold vapour laser (e.g. 628 nm, 10 W output)    -   Tunable solid state laser (e.g. 532-1060 nm), including Sd:YAG    -   Light emitting diode (LED) (e.g. 400-800 nm)    -   Diode laser (e.g. 630-850 nm, 25 W output), e.g. gallium        selenium arsenide    -   Tungsten filament lamp    -   Halogen cold light source    -   Fluorescent lamp (e.g. 10 to 30 W)        The present invention is not limited to the above-mentioned        examples of light sources, exemplary wavelengths and/or power        outputs. It is entirely possible for the present invention to be        carried out using other light sources and/or the above-mentioned        light sources with different wavelengths and/or power outputs.        The duration of exposure to the light source should be long        enough to ensure sufficient killing of the microbes. This may        vary depending on the choice of photosensitiser and light        source. For example, TBO-containing conjugates may require        exposure for between about 30 and about 45 minutes to ensure        effective killing of microbes using a 15 to 30 W fluorescent        lamp, but only about 1 to about 5 minutes using a HeNe laser. In        another embodiment, for example when the light source is of low        intensity such as exposure to natural daylight, the conjugate is        exposed to the light source for a longer period of time, such as        for several hours, for example from about 1 to about 12 hours.

The light may be delivered to the conjugate by ambient exposure, or, ifnecessary or convenient, by use of a directed means such as a fibreoptic light source or other known optical devices.

D. Antimicrobial Effect

When used as light activated antimicrobials, the conjugates as describedherein are capable of killing or inhibiting the growth ofmicroorganisms, including bacteria, viruses, fungi, protoctists andprions, that can cause disease in humans, animals or plants.

The efficacy of the conjugates as antimicrobials depends on manyfactors. The choice of nanoparticle type, choice of photosensitiser,nanoparticle size, ratio of nanoparticle:ligand:photosensitiser,concentration of photosensitiser, light source and duration of exposureto light may all influence antimicrobial activity. The skilled personcan readily determine suitable combinations.

E. Medical Applications

In one embodiment, the present invention provides conjugates as definedherein for use in treating a human or animal body by administering aneffective non-toxic amount of said conjugate, followed by exposure to asuitable light source. In particular, the present invention provides theconjugates for use in killing or preventing the growth of microbes, orfor ameliorating or reducing the incidence of proliferative celldisorders such as cancer in the human or animal body. The presentinvention also provides use of conjugates as described herein in themanufacture of a medicament for killing or preventing the growth ofmicrobes, and a method of treating a human or animal body, which methodcomprises the administration of an effective non-toxic amount of aconjugate as described herein, followed by exposure to a suitable lightsource.

In one embodiment, the conjugates of the present invention are used tokill or inhibit the growth of Staphylococcus aureus. The conjugates ofthe present invention may also be used to kill or inhibit the growth ofPropionibacterium acnes and the microbes involved in oral diseases, suchas inflammatory periodontal disease and caries, or in infections atother body sites. For example, the conjugates of the present inventionmay also be used to kill or inhibit the growth of Streptococcus sanguis,Porphyromonas gingivalis, Fusobacterium nulceatum, Actinobacillusactinomycetemcomitans, Candida albicans, Streptococcus mutans,Streptococcus pyogenes, Pseudomonas aeruginaosa, Escherichia Coli andlactobacilli.

If the conjugate comprises a targeting moiety, this may bind to themicrobes of interest, enhancing the antimicrobial effect. When thenanoparticle of such a targeted conjugate comprises core-shell particleshaving a magnetic core, it may be possible to remove the conjugates,before or after the step of exposure to a light source, by using amagnetic field. Such a step would also remove microbes attached to theconjugate via the targeting moiety, thereby “cleaning” the treated site.Such an application could be particularly advantageous when the treatedsite is a wound.

Conjugates comprising targeting moieties could also be advantageous intreating, ameliorating or reducing the incidence of proliferative celldisorders such as cancer. Thus the present invention also provides amethod of treating proliferative cell disorders such as cancer, whichmethod comprises the administration of an effective non-toxic amount ofa conjugate as described herein comprising a suitable targeting moiety,followed by exposure to a light source. Suitable light sources fortreatment of cancerous tumours have wavelengths in the near infrared(NIR) region, e.g. from about 800 to about 1600 nm. Thus, conjugatesshould be chosen such that they are active at such wavelengths: inparticular, the photosensitiser may be chosen such that it absorbs insuch a wavelength range.

In one embodiment, the conjugates as described herein are for use insystemic or topical applications. For example, the conjugates may beapplied topically to skin, wounds or a mucosal surface in order to killor inhibit the growth of microbes thereon. As a further example, theconjugates of the present invention may find application in killing orpreventing the growth of fungi, for example in infections of the nailbed. Alternatively, they may be used systemically to kill or prevent thegrowth of microbes within body tissues.

Such treatment of systemic infections may also be achieved outside thebody. For example, the present invention may comprise a method forkilling or preventing the growth of microbes in a fluid such as blood,comprising adding a conjugate as described herein to the fluid followedby exposure to a suitable light source.

The fluid containing the conjugate may be flowed into and through aphotopermeable container for irradiation, using a flow through typesystem. Alternatively, the fluid to be treated may be placed in aphotopermeable container which is agitated and exposed to the lightsource for a time sufficient to substantially inactivate the microbes,in a batch-wise type system. Any suitable apparatus may be used for sucha procedure, for example a radiation or treatment chamber. Suitablecontainers include bags, boxes, troughs, tubes or tubing. Batch-wisetreatment of the fluid may be achieved using, for example, collectionbags. Preferably, the container is agitated during treatment to mix thefluid and conjugate and ensure that the majority of the fluid is exposedto the light source. Continuous treatment may be achieved via anextracorporeal loop, wherein blood is contacted with the conjugate andexposed to a light source whilst in the loop.

The light source may be continuous or pulsed. The conjugate may be addeddirectly to the fluid to be treated, or may be flowed into thephotopermeable container separately from the fluid being treated, or maybe added to the fluid prior to placing the fluid in the photopermeabletreatment container. The conjugate may also be added to thephotopermeable container either before or after sterilization of thetreatment container.

The present invention also provides a method of disinfecting orsterilising a locus in a patient, which method comprises theadministration to the said locus of an effective non-toxic amount of aconjugate as described herein followed by exposure of said locus to asuitable light source.

The conjugates as described herein may be for use in killing orpreventing the growth of microbes in a body cavity. As noted above, bodycavity shall mean any cavity within a body such as mouth or oral cavity,nose, ear, vagina, lung, the entire digestive tract (e.g., throat,esophagus, stomach, intestines, rectum, etc.), gall bladder, bladder,any open wound or the like. The body cavity can be within a human bodyor a body of another animal. In a preferred aspect the inventionprovides the use of conjugates as described herein in the manufacture ofa medicament for use in disinfecting or sterilising tissues of a bodycavity or a wound or lesion in a body cavity by (a) contacting thetissues, wound or lesion with conjugates and (b) irradiating thetissues, wound or lesion with a suitable light source.

The wound or lesion treated may be any surgical or trauma-induced wound,a lesion caused by a disease-related microbe, or a wound or lesioninfected with such a microbe. The treatment may be applied to disinfector sterilise a wound or lesion as a routine precaution against infectionor as a specific treatment of an already diagnosed infection of a woundor lesion. In one embodiment, the body cavity is the oral cavity. Theconjugates of the present invention may also be used in other bodycavities, such as the nose, rectum, bladder, lungs, vagina, etc.

In another preferred aspect the invention provides the use of conjugatesof the present invention in the manufacture of a medicament for use inkilling or preventing the growth of disease-related microbes in a bodycavity, such as the oral cavity, nose, rectum, bladder, lungs, vagina,etc. by (a) contacting the microbes with conjugates and (b) irradiatingthe microbes with a suitable light source.

In another embodiment, the conjugates as described herein are for use inkilling or inhibiting the growth of the microbes involved in oraldiseases. Thus, when the body cavity is the oral cavity, the treatmentwith conjugates and irradiation are preferably applied to (i)destruction of disease-related microbes in a periodontal pocket in orderto treat chronic periodontitis; (ii) destruction of disease-relatedmicrobes in the region between the tooth and gingiva (gingival creviceor gingival margin) in order to treat or prevent inflammatoryperiodontal diseases, including chronic periodontitis, gingivitis andthe like; (iii) disinfection or sterilisation of drilled-out cariouslesions prior to filling; (iv) destruction of cariogenic microbes on atooth surface in order to prevent dental caries; (v) disinfection orsterilisation of dental and/or gingival tissues in other dental surgicalprocedures and (vi) treatment of oral candidiasis in AIDS patients,immunocompromised patients or patients with denture stomatitis.

For such applications, the conjugates are suitably in the form of asolution or a suspension in a pharmaceutically acceptable aqueouscarrier, but may be in the form of a solid such as a powder or a gel, anointment or a cream. The pharmaceutical composition may further compriseone or more accessory ingredients selected from buffers, salts foradjusting the tonicity of the solution, antioxidants, preservatives,gelling agents and remineralisation agents. The composition may beapplied to the infected area by painting, spreading, spraying, injectingor any other conventional technique, in order to contact the conjugatewith the microbes.

The conjugate may be left in contact with the microbes for a period oftime. The duration of time may vary depending on the particularphotosensitiser in use and the target microbes to be killed. Forexample, it can be from about 1 second to about 10 minutes. In oneembodiment, the duration of time is about 10 seconds to about 2 minutes.In another embodiment, the duration of time is about 30 seconds.

F. Non-Medical Applications

In one aspect, the present invention does not extend to the use of themixtures in methods for treatment of the human or animal body by surgeryor therapy, or in diagnostic methods practised on the human or animalbody.

The conjugates of the present invention may be used to kill or inhibitthe growth of microorganisms on inanimate objects or surfaces. In oneembodiment, the conjugates of the present invention are used to kill orinhibit the growth of Staphylococcus aureus.

The antimicrobial properties of the conjugates of the present inventionmay find application in hospitals and other places where microbiologicalcleanliness is necessary, for example food processing facilities, diningareas or play areas. Use in abattoirs is also envisaged. The conjugatesmay be applied to any suitable surface in order to sterilize ordisinfect it, for example work surfaces, wash basins, toilets, tiles,door handles or computer keyboards. In another embodiment, theconjugates may be applied to cling-film or other films or packaging,such as food packaging, for example by spraying or painting a solutionof the conjugate onto the film. Such antimicrobial films or packagingcould also be produced by incorporating the conjugate into thefilm/packaging. The cling-film type material could be wrapped around orused to cover medical/dental instruments, computer input devices, foodor drink products, surfaces etc.

The conjugates may be applied as a coating by painting, spreading orspraying and may be dried or allowed to dry naturally. They can also bemixed with a plastics material such as cellulose acetate to create anantimicrobial plastic. Such a plastics material could be used tomanufacture articles, such as computer input devices, or asantimicrobial coverings to be wrapped or coated over the surface of thearticle to be treated. Thus, in one embodiment, an article such as acomputer input device could be coated with a mixture of celluloseacetate and the conjugate.

In another embodiment, the conjugates of the present invention may beused to sterilise or disinfect textiles or fabrics. For example, theconjugates may be applied to articles such as clothes, bed sheets, labcoats, curtains or furniture. Application may be effected by, forexample, spraying or otherwise applying a suitable solution/suspensioncontaining the conjugates, or soaking in such a solution/suspension. Thearticle may then be exposed to a suitable light source for a sufficientamount of time to kill or prevent the growth of microbes in or on thearticle.

In another embodiment, the conjugates of the present invention may beused to sterilise or disinfect fluids, such as water. The presentinvention may therefore comprise a method for killing or preventing thegrowth of microbes in a fluid, such as water, comprising adding aconjugate as described herein to the fluid followed by exposure to asuitable light source. The conjugate may comprise core-shell particleshaving a magnetic core, so that it may be possible to remove theconjugates by using a magnetic field, as described above. Suchconjugates comprising magnetic particles may also comprise a targetingmoiety, which may bind to the microbes, enhancing the antimicrobialeffect and enabling the microbes to be removed along with the conjugate.

In a further embodiment, the conjugates of the present invention may beapplied to plants in order to control plant pests or pathogens such asfungi, bacteria or viruses. After application of the conjugate, forexample by spraying, the plant may be exposed to a suitable light sourcefor a sufficient amount of time to kill or prevent the growth of plantpests or pathogens. Sunlight may be such a suitable source. In oneembodiment, the conjugates of the present invention are applied tonon-edible plants.

The present invention also provides a process of killing or preventingthe growth of microbes on an inanimate object or surface, comprisingcontacting with a conjugate according to the present invention followedby exposure to a light source for a sufficient amount of time to kill orprevent the growth of microbes. Suitable light sources are describedabove. As described above, the mixture is at a suitable concentrationsuch that a desired level of antimicrobial activity is achieved at thetreatment site. For application to surfaces, the mixture may be applieddirectly by any suitable means, such as a cloth, spray or wash.

The conjugate may be left in contact with the microbes for a period oftime, such as those described above for medical applications.

III. EXAMPLES

please note that these examples are for the purpose of illustration onlyand are not to be construed as limiting the scope of the invention inany way.

Example 1

Gold nanoparticles (2.0 nm diameter; British Biocell International) inwater (15×10¹³ particles per ml) were mixed with an equal volume of anaqueous solution of toluidine blue O (40 μM) and left at roomtemperature for 15 minutes. 100 μl of the gold-TB solution was added to100 μl of a suspension of Staphylococcus aureus NCTC 6571 in phosphatebuffered saline (PBS) and this was irradiated with white light from afluorescent white lamp for 10 minutes. Controls consisted of:

(i) TB (final concentration=10 μM) and bacteria, irradiated for the sameperiod of time,

(ii) nanogold (diluted 1:1 with water) and bacteria, irradiated for thesame period of time,

(iii) bacteria without TB or nanogold, not irradiated (“control”).

After irradiation, the number of surviving bacteria was determined byviable counting. The results of the experiments (carried out twice withduplicate counts on each occasion) are shown in Table 1. The goldnanoparticles alone when irradiated did not achieve significant killingof the bacteria. The TB-gold achieved approximately a one log greaterkill than the TB alone—99.3% kill as opposed to a 93.7% kill. Note thatthe TB concentration and light energy dose used were designed to givesub-optimal kills so that differences in efficacy of the TB and theTB-nanogold could be discerned. Preliminary experiments using 30 minuteslight exposure achieved total kills of the bacterial suspensions in bothcases. TABLE 1 Sample¹ S. aureus (cfu/ml) % Kill Control 135000000 —Gold only 81000000 40.0 TB only 8570000 93.7 Mixture 983000 99.3 (L +TB + G +)¹Samples were irradiated with light from a 28 W fluorescent white lamp.US application number 60/821,423 mentions an 18 W lamp.

Example 2

Production of Water-Soluble Gold Nanoparticles

HAuCl₄.3H₂O (42 mg, 0.11 mmol) was dissolved in deionised water (25 ml)to form solution A (˜5 mM). Na₃C₆H₅O₇.2H₂O (125 mg, 0.43 mmol) wasdissolved in deionised water (25 ml) to give solution B (˜20 mM).Solution A (1 ml) was stirred with deionised water (18 ml) and boiledfor 2 min. Then solution B (1 ml) was added dropwise over a period ofapproximately 50 sec. causing the colour change from clear to blue topink/purple. After a further 1 min. of heating, the solution was left tocool to room temperature. Two batches of nanogold particles were usedfor subsequent antibacterial assays—these are designated NN1 and NN2.The absorption spectrum of NN2 showed the wavelength of maximumabsorption, λ_(max) to be 527 nm. Batch NN1 had a λ_(max) of 522 nm.Particle size analysis (position of UV plasmon absorption band measuredusing transmission electron microscope) of batch NN1 gave an averagediameter of 14.76±2.34 nm.

Effect of Concentration of Photosensitiser

Gold nanoparticles of approximately 15 nm in diameter (batches NN1 andNN2 above) were mixed with an equal volume of aqueous toluidine blue O(TB) and left at room temperature for 15 minutes. TB was used at a finalconcentration of 1, 5, 10, 20 or 50 μM. 100 μl of the TB-gold mixturewas added to 100 μl of a suspension of Staphylococcus aureus NCTC 6571in phosphate buffered saline (PBS) (adjusted to an optical density of0.05), and samples were irradiated with a fluorescent white light (28 W)for 10 minutes. S. aureus+TB only, and S. aureus+PBS, withoutphotosensitiser or nanogold were used as controls. The finalconcentration of nanogold used was 1×10¹⁵ particles/ml. Afterirradiation, the numbers of surviving bacteria were enumerated by viablecounting. The results are shown in Table 2 below.

In the case of the 15 nm nanogold, there was little enhancement oflethal photosensitisation (compared with that achieved when TB was usedin the absence of nanogold) when the TB concentration was 1 μM whereasenhancement was evident using higher TB concentrations of 5, 10 and 20μM. Enhancement was greatest using 10 and 20 μM TB. Enhancement appearsto be dependent on the ratio of TB to nanogold. There was littleenhancement of lethal photosensitisation when the TB concentration was10 or 100 μM, whereas enhancement was greatest using TB concentrationsof 20 and 50 μM.

Example 3

The method of Example 2 was repeated using gold nanoparticles of 2 nmdiameter (British Biocell International). The final concentration ofnanogold used was 4×10¹³ particles/ml. TB was used at a finalconcentration of 10, 20 or 50 μM. The results are shown in Table 2below. When the 2 nm nanogold particles were used, enhancement of lethalphotosensitisation was evident using 20 μM TB but not when either 10 μMor 50 μM TB was used.

Example 4

Effect of Concentration of Gold Nanoparticles

Experiments were performed as for Example 3, with the followingmodifications. Prior to mixing with the photosensitiser, the goldnanoparticles were either left undiluted, or diluted 1 in 10 or 1 in 100in sterile, distilled water. The nanoparticles were then added to TB(final concentration 20 μM). The samples were then illuminated for 30seconds using a fibre optic white light source (Schott KL200). Thesurviving bacteria were enumerated by viable counting as before. Theresults are shown in Table 2 below. When the nanoparticles were diluted1 in 10 a greater enhancement was achieved compared with that obtainedusing undiluted nanogold.

Example 5

Example 4 was repeated using methylene blue (MB; 20 μM) as thephotosensitiser. The results are shown in Table 2 below. The enhancementachieved by the nanogold with a larger particle size (15 nm) was notincreased when the nanogold concentration was decreased.

Example 6

Example 5 was repeated using 2 nm gold nanoparticles (British BiocellInternational). The results are shown in Table 2 below. Diluting the 2nm gold nanoparticles enhanced the killing of S. aureus slightly whenused in combination with methylene blue.

Example 7

Example 6 was repeated using tin chlorin e6 (SnCe6; 20 μg/ml) as thephotosensitiser. The illumination time was 10 minutes. The results areshown in Table 2 below. Diluting the 2 nm gold nanoparticles enhancedthe killing of S. aureus when used in combination with tin chlorin e6.

Example 8

Example 3 was repeated using nile blue sulphate as the photosensitiser.Samples were illuminated for 30 minutes. The results are shown in Table2 below. TABLE 2 Concentration of Nanoparticle photosensitiser¹Nanoparticle concentration¹ Example Photosensitiser (μM) size (nm)(particles/ml) Result² 2 Toluidine blue  1 15 1 × 10¹⁵ —  5 * 10 **/***20 **** 50 **** 100  ** 3 Toluidine blue 10 2 4 × 10¹³ * 20 **** 50 * 4Toluidine blue 20 15 1 × 10¹⁵ *** 1 × 10¹⁴ **** 5 Methylene blue 20 15 1× 10¹⁵ **** 1 × 10¹⁴ **** 1 × 10¹³ **** 6 Methylene blue 20 2 4 × 10¹³**** 4 × 10¹² **** 4 × 10¹¹ **** 7 Tin chlorin e6  20³ 2 4 × 10¹³ — 4 ×10¹² ** 4 × 10¹¹ *** 8 Nile blue sulphate 10 2 4 × 10¹³ *** 20 **** 50****¹concentration in mixed solution²Key: — less than 50% kill; * 50-90% kill; ** 90-95% kill; *** 95-99%kill; **** 99-100% kill³concentration in μ/ml

Example 9

Synthesis of TBO-Tiopronin-Gold Nanoparticle Conjugates

Chemicals: Hydrogen tetrachloroaurate (tetrachloroauric acid;HAuCl₄.3H₂O, 99.99%), N-(2-mercaptopropionyl)glycine (tiopronin, 99%)and sodium borohydride (99%) were purchased from Aldrich. Toluidine BlueO (“TBO”, 90%) was purchased from Acros Organics. Buffers were preparedaccording to standard laboratory procedure. All other chemicals werereagent grade and used as received. The synthesis of the conjugatesinvolved two steps:

(1) Synthesis of tiopronin-gold nanoparticle conjugate; and

(2) Preparation of TBO-tiopronin-gold nanoparticle conjugate.

Synthesis of Tiopronin-Gold Nanoparticle Conjugate:

Tetrachloroauric acid (0.62 g; 1.57 mmol) and N-(2mercaptopropionyl)glycine (tiopronin, 0.77 g; 4.72 mmol) were dissolvedin 6:1 methanol/acetic acid (70 mL) giving a ruby red solution. Sodiumborohydride (NaBH4, 1.21 g; 32 mmol) in water (30 mL) was added withrapid stirring, whereupon the solution temperature immediately rose from24° C. (room temperature) to 44° C. (returning to room temperature inca. 15 min). Meanwhile, the solution pH increased from its initial 1.2value to 5.1. The black suspension that was formed was stirred for anadditional 30 min after cooling, and the solvent was then removed undervacuum at ≦40° C.

The crude reaction product was completely insoluble in methanol butquite soluble in water. It was purified by dialysis, in which the pH ofthe crude product dissolved in water (80 mL) was adjusted to 1 bydropwise addition of concentrated hydrochloric acid (HCl). This solutionwas loaded into 20 cm segments of cellulose ester dialysis membrane(Spectra/Por CE, MWCO=12000), placed in a 4 L beaker of water, andstirred slowly, recharging with fresh water ca. every 12 hours over thecourse of 72 hours. The dark tiopronin-gold nanoparticle conjugatesolution was collected from the dialysis tube, and the solvent wasremoved by freeze-drying. The product materials were found to bespectroscopically clean (¹H NMR in D₂O, 10 mg of sample: absence ofsignals due to unreacted thiol or disulfide and acetate byproducts).Elemental analysis of the dialysed tiopronin-gold nanoparticle conjugategave the following. Anal. Found: C, 11.70; H, 1.65; N, 2.55; S, 5.73.Calcd for C₄₂₅H₆₈₀O₂₅₅N₈₅S₈₅Au₂₀₁: C, 9.56; H, 1.28; N, 2.23; O, 7.65;S, 5.11; Au, 74.17.

Preparation of TBO-Tiopronin-Gold Nanoparticle Conjugate

Tiopronin-gold nanoparticle conjugates (MW=53376.38 g/mol, 100 mg, 1.87μmol) were dissolved in 50 mM 2-(N-morpholino)ethanesulfonic acid (MES)buffer (pH 6.5; 30 mL) and the solution then made up to 0.1 M in1-[3-(dimethylamino)-propyl]-3-ethylcarbodiimide hydrochloride (EDC) and5.31 mM in N-hydroxysulfosuccinimide sodium salt. Toluidine Blue O (TBO,61 mg, 0.2 mmol) was added, and the solution was stirred for 24 hours.Then, the reaction mixture was dialyzed as described above for 144hours. The dark purple TBO-tiopronin-gold nanoparticle conjugatesolution was collected from the dialysis tube, and the solvent wasremoved by freeze-drying. ¹H NMR spectroscopy (in D₂O/phosphatebuffer-d; 8 mg of sample) revealed pure product. The number of moleculesof TBO coupled to each nanoparticle was 15.4, as determined by ¹H NMR.This value was verified by elemental analysis. Anal. Found: C, 14.45; H,1.91; Cl, 0.86; N, 3.35; S, 5.58. Calcd forC₆₅₆H_(895.6)Cl_(15.4)O_(239.6)N_(131.2)S_(100.4)Au₂₀₁: C, 13.63; H,1.56; Cl, 0.94; N, 3.18; O, 6.63; S, 5.57; Au, 68.49.

The following Examples 10-13 deal with lethal photosensitisation ofStaphylococcus aureus using a TBO-tiopronin-gold nanoparticle conjugate.

Example 10 White Light

An overnight culture of Staphylococcus aureus NCTC 6571 (1 ml; grownaerobically at 37° C., with shaking, in Nutrient Broth no. 2) wascentrifuged and the pellet resuspended in phosphate buffered saline(“PBS”, 1 ml). The optical density at 600 nm was adjusted to 0.05 inPBS, in order to give an inoculum of approximately 10⁷-10⁸ cfu/ml. ATBO-tiopronin-gold nanoparticle conjugate, prepared by a methodanalogous to that described in Example 9, approximate compositionAu₂₀₁tiopronin₈₅TBO₁₁, was suspended in sterile distilled water at aconcentration of 4.6 mg/ml. The conjugate solution was then diluted 1 in2, 1 in 10 and 1 in 100 in sterile distilled water. In a 96-well plate,50 μl aliquots of the conjugate were added to 50 μl of the bacterialsuspension, in triplicate, and irradiated with white light (28 W compactfluorescent lamp; 3600±20 lux) for 35 minutes. Controls consisted of:

-   -   (i) bacteria without conjugate, kept in the dark for an equal        amount of time (“control”);    -   (ii) bacteria with conjugate, kept in the dark for an equal        amount of time;    -   (iii) irradiated tiopronin-gold nanoparticle conjugate with free        TBO ; and    -   (iv) irradiated tiopronin-gold nanoparticle conjugate alone.        After irradiation, samples were serially diluted 1 in 10 to a        dilution factor of 10⁻⁴ and spread in duplicate onto 5% horse        blood agar plates. The plates were then incubated aerobically at        37° C. for approximately 48 hours. After incubation, the        surviving cfu/ml was calculated. The results are summarised in        Table 3. The conjugate had no effect when irradiated with white        light for 35 minutes when used neat or at a dilution of 1 in 2,        and little effect at a dilution of 1 in 100. However,        antibacterial activity (approximately 4 log reduction in colony        forming units/ml) was observed when the conjugate was diluted 1        in 10.

The absence of killing by the undiluted and 1 in 2 dilutions of theconjugate were likely to be due to light absorption by the very darklycoloured solutions. The small kills detected using a 1 in 100 dilutionwere probably due to the very low concentrations of TBO present. Whennot exposed to white light, no antibacterial activity was seen at anyconcentration of the conjugate tested. Furthermore, neither free TBO incombination with the tiopronin-gold nanoparticles, nor thetiopronin-gold nanoparticles alone achieved any killing of S. aureus6571 at any of the concentrations tested.

Example 11 HeNe Laser

The method of Example 10 was repeated using a helium-neon laser (poweroutput=35 mW; emitting light at 632 nm) instead of white light, with anirradiation time of one minute. The results are shown in Table 3. Aswith the white light, the concentration that achieved the best killingof S. aureus was a 1 in 10 dilution. However in contrast to the resultsusing the white light; antibacterial activity (approximately 2 logreduction in cfu/ml) was also observed when the conjugate was diluted 1in 2.

Example 12 Effect of Light Dose (White Light)

The method of Example 10 was repeated, using TBO-Tiopronin-goldnanoparticle conjugate at 1 in 10 dilution. Samples were illuminatedwith the same white light source as described above for 15, 30, or 45minutes. Results are shown in Table 3. No antibacterial effect wasobserved after 15 minutes. The conjugate achieved approximately a twolog reduction in the surviving cfu/ml after 30 minutes irradiation,increasing to an approximately 5 log reduction in cfu/ml after 45minutes. The effect of TBO alone was also investigated, and was found tohave no effect when irradiated with white light for any length of time.

Example 13 Effect of Light Dose (HeNe Laser)

The method of Example 12 was repeated, but samples were irradiated withthe HeNe laser described in Example 3 for 0.5, 1, 1.5, 2 or 5 min.Results are shown in Table 3. This was then repeated with irradiationfor one, two or five minutes. Highly effective killing was achieved forexposure times of 1 min and above. As seen with white light, the resultsshowed a dose response, in which killing of S. aureus increased withincreased irradiation time, with most killing being seen at five minutes(approximately 5.5 log reduction in cfu/ml). TABLE 3 Dilution of LightIrradiation conjugate Example Source time (min) solution¹ Result² 10White 35 Neat — 1 in 2 — 1 in 10 **** 1 in 100 ** 11 HeNe laser 1 Neat —1 in 2 **** 1 in 10 **** 1 in 100 * 12 White 15 1 in 10 — 30 **** 45**** 13 HeNe laser 0.5 1 in 10 *** 1 **** 1.5 **** 2 **** 5 ****¹Before mixing with bacterial suspension²Key: — less than 50% kill; * 50-90% kill; ** 90-95% kill; *** 95-99%kill; **** 99-100% kill

Examples 14-15 deal with lethal photosensitisation of Staphylococcusaureus using a different TBO-tiopronin-gold nanoparticle conjugate.

Example 14 White Light

An overnight culture of Staphylococcus aureus NCTC 6571 (1 ml; grownaerobically at 37° C., with shaking, in Nutrient Broth no. 2) wascentrifuged and the pellet resuspended in phosphate buffered saline(“PBS”, 1 ml). The optical density at 600 nm was adjusted to 0.05 inPBS, in order to give an inoculum of approximately 10⁷-10⁸ cfu/ml. TheTBO-tiopronin-gold nanoparticle conjugate prepared in Example 1,approximate composition Au₂₀₁tiopronin₈₅TBO_(15.4), was suspended in PBSat a concentration of 4.6 mg/ml, such that the final TBO content wasapproximately 1 mM. The conjugate solution was then diluted in PBS togive final TBO concentrations of approximately 2 μM, 1.0 μM, 0.5 μM and0.25 μM. In a 96-well plate, 50 μl aliquots of the conjugate were addedto 50 μl of the bacterial suspension, in triplicate, and irradiated withwhite light (28 W compact fluorescent lamp; 3600±20 lux) for 30 minutes.

Controls consisted of:

-   -   (i) bacteria without conjugate;    -   (ii) TBO;    -   (iii) irradiated tiopronin-gold nanoparticle conjugate with free        TBO at a final TBO concentration of 1 μM; and    -   (iv) irradiated tiopronin-gold nanoparticle conjugate alone: it        was calculated that prior to dilution, the TBO-tiopronin-gold        nanoparticle conjugate contained approximately 81 μM        tiopronin-gold, and therefore a stock solution of the        tiopronin-gold nanoparticle conjugate was made up to this        concentration and then diluted accordingly.        After irradiation, samples were serially diluted 1 in 10 to a        dilution factor of 10⁻⁴ and spread in duplicate onto 5% horse        blood agar plates. The plates were then incubated aerobically at        37° C. for approximately 48 hours. After incubation, the        surviving cfu/ml was calculated. The results are shown in FIG. 1        and summarised in Table 4. FIG. 1 shows the effect of TBO and        the TBO-tiopronin-gold nanoparticle conjugate on viability of        Staphylococcus aureus 6571 following exposure to white light for        30 minutes, or incubation in the dark with TBO or the        TBO-tiopronin-gold nanoparticle conjugate. The white bar (□) in        FIG. 1 denotes the viable count of the original bacterial        suspension, and the dotted bar (        ) represents the viable count of the bacterial suspension after        exposure to white light alone. The diagonal stripe bar (        ) represents the viable count of the bacterial suspension after        incubation in the dark with TBO. The horizontal strip bar (        ) represents the viable count of the bacterial suspension after        incubation in the dark with the TBO-tiopronin-gold nanoparticle        conjugate. The grey bar (        ) represents the viable count of the bacterial suspension after        TBO and exposure to white light. The black bar (▪) represents        the viable count of the bacterial suspension after        TBO-tiopronin-gold nanoparticle conjugate and exposure to white        light. There was a concentration-dependent reduction in the        viable count of S. aureus on irradiation with white light for 30        mins. At a concentration of 2.0 μm, an approximately 5.5 log₁₀        reduction in the viable count was observed. Substantial kills        were achieved using a conjugate concentration as low as 0.5 μm,        whereas free TBO exhibited significant kills of the organism        only at a concentration of 2.0 μm. The TBO-free tiopronin-gold        nanoparticles did not achieve any killing of S. aureus 6571 at        any of the concentrations tested. Mixtures of various ratios of        the tiopronin-gold conjugate and a sub-optimal concentration of        TBO (1.0 μM) did not result in killing of the S. aureus on        irradiation with white light.

Example 15 HeNe Laser

The method of Example 14 was repeated using a helium-neon laser (poweroutput=35 mW; emitting light at 632 nm) instead of white light, with anirradiation time of one minute. The results are shown in FIG. 2 andTable 4. FIG. 2 shows the effect of TBO and the TBO-tiopronin-goldnanoparticle conjugate on viability of S. aureus 6571 following exposureto HeNe laser light for 1 minute, or incubation in the dark with TBO orthe TBO-tiopronin-gold nanoparticle conjugate. The white bar (□) in FIG.2 denotes the viable count of the original bacterial suspension, and thedotted bar (

) represents the viable count of the bacterial suspension after exposureto HeNe laser light alone. The diagonal stripe bar (

) represents the viable count of the bacterial suspension afterincubation in the dark with TBO. The horizontal strip bar (

) represents the viable count of the bacterial suspension afterincubation in the dark with the TBO-tiopronin-gold nanoparticleconjugate. The grey bar (

) represents the viable count of the bacterial suspension after TBO andexposure to HeNe laser light. The black bar (▪) represents the viablecount of the bacterial suspension after TBO-tiopronin-gold nanoparticleconjugate and exposure to HeNe laser light. As with the white light, thekills achieved were concentration-dependent—significant kills wereachieved when the conjugate was used at a concentration as low as 0.5μM. TABLE 4 TBO Irradiation time concentration Example Light Source(min) (μM) Result¹ 14 White 30 2.0 **** 1.0 **** 0.5 **** 0.25 * 15 HeNelaser 1 2.0 **** 1.0 **** 0.5 * 0.25 —¹Key: — less than 50% kill; * 50-90% kill; ** 90-95% kill; *** 95-99%kill; **** 99-100% kill.

The preferred embodiments of the present invention have been disclosed.A person of ordinary skill in the art would realize however, thatcertain modifications would come within the teachings of this invention.Therefore, the following claims should be studied to determine the truescope and content of the invention.

1. A method of killing or preventing the growth of microbes comprising:applying a metallic nanoparticle-ligand-photosensitiser conjugate to adesired treatment site, wherein: ligand of the conjugate is awater-solubilising ligand; metallic nanoparticle and photosensitiser ofthe conjugate are chosen such that the conjugate generates singletoxygen and/or free radicals; and the conjugate acts as a light-activatedantimicrobial.
 2. The method of claim 1 further comprising irradiatingthe conjugate at the treatment site.
 3. The method of claim 1, whereinthe metallic nanoparticle has a diameter of from about 1 nm to about 30nm.
 4. The method of claim 1, wherein the metallic nanoparticlecomprises at least one metal selected from a group consisting of gold,silver, and copper.
 5. The method of claim 1, wherein the metallicnanoparticle is selected from a group consisting of an alloy of gold andsilver, an alloy of gold and copper, an alloy of gold, silver, andcopper, and an alloy of gold, silver, and aluminium.
 6. The method ofclaim 1, wherein the metallic nanoparticle comprises core-shellparticles.
 7. The method of claim 6, wherein the core-shell particlescomprise a magnetic core or magnetic layer.
 8. The method of claim 1,wherein the conjugate further comprises at least one targeting moiety.9. The method of claim 1, wherein the ligand comprises a compoundselected a group consisting of thiol, xanthate, disulfide, dithiol,trithiol, thioether, polythioether, tetradentate thioether,dithiocarbamate, phosphine, phosphine oxide, alkanolamine, aminoacid,carboxylate, isocyanide, acetone, iodine, dialkyl-diselenide,thioaldehyde, thion acid, thion ester, thioamide, thioacyl halide,sulfoxide, sulfenic acid, sulfenyl halide, isothiocyanate, isothiourea,aliphatic or aromatic selenol, selenide, diselenide, selenoxide,selenenic acid, selenenyl, aliphatic or aromatic tellurol, telluride,and ditelluride.
 10. The method of claim 1, wherein the ligand comprisesa compound selected from the group consisting of 3-mercaptopropionicacid, 4-mercaptobutyric acid, 3-mercapto-1,2-propanediol, cysteine,methionine, thiomalate, 2-mecaptobenzoic acid, 3-mercaptobenzoic acid,4-mecaptobenzoic acid, tiopronin, selenomethionine,1-thio-beta-D-glucose, glutathione and ITCAE pentapeptide.
 11. Themethod of claim 1, wherein the photosensitiser is selected from a groupconsisting of porphyrin, phthalocyanine, chlorin, bacteriochlorin,phenothiazinium, phenazines, acridine, texaphyrin, cyanine,anthracyclin, pheophorbide, sapphyrin, fullerene, halogenated xanthene,perylenequinonoid pigment, gilvocarcin, terthiophene,benzophenanthridine, psoralen, riboflavin, arianor steel blue, tryptanblue, crystal violet, azure blue cert, azure B chloride, azure 2, azureA chloride, azure B tetrafluoroborate, thionin, azure A eosinate, azureB eosinate, azure mix sicc and azure II eosinate.
 12. The method ofclaim 1, wherein the conjugate is in aqueous solution.
 13. The method ofclaim 1, wherein an effective non-toxic amount of the conjugate isapplied to the treatment site during the applying step.
 14. The methodof claim 1, wherein the method is used to ameliorate or reduce incidenceof proliferative cell disorders such as cancer.
 15. The method of claim1, wherein the microbes are selected from a group consisting ofStaphylococcus aureus, Propionibacterium acnes, Streptococcus sanguis,Porphyromonas gingivalis, Fusobacterium nulceatum, Actinobacillusactinomycetemcomitans, Candida albicans, Streptococcus mutans,lactobacilli, and a combination thereof.
 16. The method of claim 1,wherein the microbes are Methicillin-Resistant Staphylococcus aureus.17. The method of claim 2, wherein the irradiating step is achieved by alight source selected from the group consisting of fluorescent, laser,light emitting diode(s), halogen, incandescent, chemiluminescent, and acombination thereof.
 18. The method of claim 2, wherein the irradiatingstep is achieved by a laser.
 19. The method of claim 18, wherein thelaser is a HeNe laser.
 20. The method of claim 2, wherein theirradiating step is achieved by white light.
 21. The method of claim 1,wherein the treatment site is selected from a group consisting of a bodycavity, skin, mucosal surface, nail bed, and an inanimate object. 22.The method of claim 1, wherein the treatment site is a body cavity. 23.The method of claim 22, wherein the body cavity is an oral cavity. 24.The method of claim 1, wherein the treatment site is skin.
 25. Themethod of claim 1, wherein the treatment site is surface of an inanimateobject.
 26. The method of claim 1, wherein the method is used to treatperiodontitis.
 27. The method of claim 1, wherein the method is used totreat gingivitis.
 28. The method of claim 1, wherein the method is usedto disinfect or sterilize the treatment site.
 29. The method of claim 8,wherein the method is used to ameliorate or reduce the incidence ofproliferative cell disorders.
 30. The method of claim 1, whereinmetallic nanoparticle of the conjugate comprises gold, ligand of theconjugate comprises tiopronin, and photosensitiser of the conjugatecomprises toluidine blue O.
 31. The method of claim 2, wherein theconjugate comprises core-shell particles having a magnetic core andfurther comprises at least one targeting moiety, and the method furthercomprises a step of removing the conjugates from the treatment siteusing a magnetic field before or after the irradiating step, therebyalso removing targeted microbes.
 32. A metallicnanoparticle-ligand-photosensitiser conjugate, wherein metallicnanoparticle of the conjugate comprises gold, ligand of the conjugatecomprises tiopronin, and photosensitiser of the conjugate comprisestoluidine blue O.
 33. The conjugate of claim 33, wherein the conjugatecomprises from about 5 to about 20 toluidine blue O groups pernanoparticle-ligand core.
 34. A composition comprising the conjugate ofclaim
 32. 35. The composition of claim 34 wherein the conjugate issuspended in an aqueous solution.
 36. A method of producing a metallicnanoparticle-ligand-photosensitiser conjugate comprising: Providing ametallic nanoparticle-ligand core comprising a metallic nanoparticlehaving bonded thereto at least one ligand having first and secondfunctional groups, wherein the ligand is bonded to the metallicnanoparticle via the first functional group; and Reacting the secondfunctional group of the at least one ligand with a functional group of aphotosensitiser.
 37. The method of claim 36, wherein the providing stepand the reacting step are carried out in aqueous solution.